The bifunctional modification reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) has been used to link two proteins together through a disulfide bond. The reagent is reacted with the first protein to introduce an active disulfide-containing group in the modification step. A second protein, which contains a free thiol group, is then added to form a disulfide bond between the two proteins in the conjugation step. Many derivatives of SPDP and imide versions of SPDP have been described (U.S. Pat. No. 4,563,304; J. Carlsson et al. 173 Biochem. J. 723-737 (1978); Goff D. A., Carroll, S. F. 1 BioConjugate Chem. 381-386 (1990); L. Delprino et al. 82 J. Pharm. Sci. 506-512 (1993); S. Arpicco et al., 8 BioConjugate Chem 327-337 (1997)).
Conjugates of cell binding agents with highly cytotoxic drugs have been described (U.S. Pat. Nos. 5,208,020 and 5,416,064; R. V. J. Chari et al., 52 Cancer Res. 127-131 (1992). In these conjugates, the cell binding agents are first modified with a bifunctional agent such as SPDP to introduce an active disulfide moiety. Reaction with a thiol-containing cytotoxic drug provides a conjugate in which the cell binding agent, such as a monoclonal antibody, and drug are linked via disulfide bonds. In order to enhance the in vivo stability of this disulfide link, it is important to provide sterically hindered disulfide bonds. This objective can be achieved by using cross-linkers that bear one or two methyl substituents on the carbon atom geminal to the disulfide bond or by using drugs bearing one or two methyl substituents on the carbon atom bearing the thiol substituent. However, introduction of such hindered disulfide bonds on cell binding agents or hindered thiols on the drugs results in a marked decrease in the rate of reaction of the thiol-containing drug and the cell binding agent. Thus, processes for the production of conjugates become either impossible, or time consuming and uneconomical. In addition, the extended reaction time causes unwanted dimerization of the thiol-containing drug and consequent loss of reactivity and low yields of product. In the case of monoclonal antibodies and fragments thereof, slow reaction rates of disulfide exchange between the hindered disulfide bond and the thiol-containing drug leads to the undesired side reaction of disulfide bond scission between the heavy and light chains of the antibody or fragment.
Thus there is a need to provide cross-linkers that will provide for an accelerated disulfide exchange reaction rate between the modified cell binding agent and the thiol substituent on the cytotoxic drug. In addition, since cell binding agents, such as monoclonal antibodies, are only soluble in aqueous solutions, it is also desirable to provide cross-linkers that are soluble in water or require only a small percentage (<5% v/v) of an organic solvent to maintain solubility in aqueous solutions.